Prevention of experimental allergic encephalomyelitis by nonencephalitogenic basic peptides

The biological properties of the chymotrypsin-treated encephalitogenic basic protein are described. The basic protein, isolated from bovine spinal cord, was digested with chymotrypsin and filtered through Sephadex gel resulting in three distinct well-separated peaks starting at the void volume of the column. Tubes common to each peak were combined into Fractions I, II, and III, respectively. More than 90% of the original protein was recovered in the three fractions. Fraction II, representing 76% of the original protein, was nonencephalitogenic when tested in guinea pigs at 0.010-, 0.025-, and 0.500-mg doses emulsified in complete Freund's adjuvant. Guinea pigs immunized with Fraction II were protected from EAE when challenged with encephalitogenic emulsions. A critical dose of 1.0 mg completely protected the animals from disease, while partial protection was obtained with lower doses. In addition to producing circulating antibodies, animals sensitized with Fraction II, the encephalitogenic tryptophan peptide or the basic protein displayed a delayed-type hypersensitivity response when skin tested with either of the three antigens. The positive skin reactivity in animals sensitized with Fraction II was not followed by EAE during 5 months of observation. In contrast, animals sensitized with extracts from bovine tissues other than the central nervous system were not protected from disease when challenged with encephalitogenic emulsions.The main finding here reported is the prevention of EAE with nonencephalitogenic peptides derived from the parent EAE-producing protein. The peptides retain the ability to induce delayed-type hypersensitivity and provide antigens to study the role of delayed hypersensitivity in experimental allergic encephalomyelitis.     

Myelin lipophilin-induced demyelinating disease of the central nervous system

Purified lipophilin, a hydrophobic lipoprotein of myelin, induces a cell-mediated demyelinating disease of the central nervous system similar to experimental allergic encephalomyelitis (EAE) induced by the myelin basic protein (MBP). Guinea pigs challenged with lipophilin (emulsified with CFA) developed clinical and histological signs of disease indistinguishable from those developed by animals similarly challenged with MBP. Both lipophilin and MBP induced and elicited delayed-type hypersensitivity in animals challenged with respective antigens. Tryptophan, an essential component of the MBP-determinant for disease in guinea pigs, is required for the encephalitogenicity of lipophilin.     

Experimental allergic encephalomyelitis in Lewis rats: inhibition by bacterial lipopolysaccharides and acquired resistance to reinduction by challenge with myelin basic protein

In 2-mo-old Lewis rats immunized with bacterial lipopolysaccharides (LPS) precomplexed to guinea pig myelin basic protein (BP), the clinical and histologic manifestations of experimental allergic encephalomyelitis (EAE) were diminished compared with BP-treated controls. Similarly, in animals immunized with BP and challenged with BP-LPS at the same time or as long as 5 days after, the immunization with BP also inhibited the disease. That this capacity to reduce the incidence of BP-induced EAE is a unique property of LPS was suggested by the fact that other negatively charged molecules, such as DNA, RNA, and dextran sulphate, were not effective in inhibiting the clinical signs of EAE. After recovery from EAE induced by BP, some animals develop a recurrence of the disease if challenged with BP at appropriate intervals. However, after recovery from mild EAE induced by BP-LPS and after challenges with EAE-initiating BP antigens, secondary EAE was inhibited significantly.     

Successful immunization against experimental allergic encephalomyelitis with myelin basic protein-sensitized allogeneic lymphocytes

Prevention and suppression of experimental allergic encephalomyelitis were demonstrated in rats, guinea pigs, and rabbits immunized with allogeneic, but not with syngeneic lymphocytes from susceptible donors sensitized to myelin basic protein (MBP). Donor lymphnode, splenic, or peripheral blood lymphocytes were effective in inducing a state of unresponsiveness to an encephalitogenic challenge in either of the three species. Unresponsiveness was not obtained in recipients immunized with sensitized allogenic lymphocytes and simulatenously challenged with MBP suggesting that a time lapse between immunization and challenge is necessary for the development of protective immunity. Induced in immunized recipients, unresponsiveness was transferred into normal syngeneic recipients with immunoglobulin-G (IgG) isolated from protected donors before challenge. Furthermore, both immunized and IgG recipients failed to develop cell-mediated immunity after challenge with MBP. The results show that prevention and suppression of EAE was mediated by antibodies which inhibited the development of delayed type hypersensitivity to the challenging antigen.     

Immunomorphological characteristics of experimental allergic encephalomyelitis induced by an encephalitogenic polypeptide

Encephalitogenic, immunogenic properties of the polypeptide fraction of myelin basic protein (FBP) and CNS lesions have been examined in animals with experimental allergic encephalomyelitis (EAE). FBP was isolated from bovine spinal cord using column chromatography. Administration of 1.0 or 0.1 microgram FBP mixed with complete Freund adjuvant caused neurological and histological EAE manifestations in 76% and 26% of guinea-pigs, respectively. Circulating anti-FBP antibodies were not found in sensitized animals, whereas the incidence and intensity of skin reaction of delayed type hypersensitivity to FBP correlated with the development of EAE and the onset of the disease. Perivascular cell infiltration and demyelination noted in the spinal cord and brain of guinea-pigs were similar to those observed after inoculation of the brain white matter or brain tissue homogenate.     

Experimental allergic encephalomyelitis: successful treatment in vivo with a monoclonal antibody that recognizes T helper cells

Monoclonal antibody W3/25, which recognizes rat T helper cells, was injected i.p. or i.v. into Lewis rats with clinical signs of experimental allergic encephalomyelitis (EAE). This treatment, which was given 12 or 13 days after inducing EAE by the injection of purified myelin basic protein, significantly affected the course of the disease in that treated animals recovered, on the average, in 2 days, whereas control animals exhibited signs of EAE for a minimum of 4 days. Two pan-T cell monoclonal antibodies given in similar dosage failed to affect the course of the disease.     

The influence of cyclosporin A on the development of actively induced and passively transferred experimental allergic encephalomyelitis

The immunosuppressive effects of cyclosporin A (CsA) were tested on actively induced and passively transferred experimental allergic encephalomyelitis (EAE). Actively induced EAE could be inhibited if CsA was administered per os at 25 mg/kg/day but not at 10 mg/kg/day. Passive transfer of clinical EAE occurred in all cell recipients including those fed CsA at either 25 or 50 mg/kg/day. Cyclosporin A could inhibit the development of transfer active cells in vitro and in vivo, however, inhibition of transfer active populations by CsA required the presence of CsA during the initial stage of cell response. If CsA was added to Con A-stimulated spleen cell cultures after a delay of 24 hr then these cells transferred clinical disease. Similarly, animals fed CsA concurrently with basic protein sensitization did not develop cell populations capable of transferring EAE. If CsA feeding commenced 2 or 4 days following sensitization all basic proteinsensitized animals still failed to develop EAE; however these latter groups of animals were a suitable source of cells capable of transferring some signs of clinical EAE.     

Effect of levamisole and etimisole on the development of experimental allergic encephalomeylitis

A single subcutaneous inoculation with 0.02 mg of heterologous myelin basic protein (MBP) in combination with Freund's complete adjuvant resulted in clinical and histological manifestations of experimental allergic encephalomyelitis (EAE) in 80-90% of treated guinea pigs. Daily parenteral administration of levamisole and etimisole during the latent period produced a suppressive effect on EAE development, reducing morbidity and mortality rates and preventing pathomorphological changes in the CNS. Animals receiving the drugs had decreased delayed hypersensitivity reactions to MBP in vitro. Etimisole brought about a moderate decline in the formation of circulating anti-MBP antibodies, while levamisole did not affect the strength of the humoral response, something which confirmed the primary role of cell-mediated immune reactions in the CNS demyelinization process. The reported findings may be significant in developing therapeutic strategies with respect to demyelinization diseases.     

Evolutionary Divergence in the Structure of Myelin Basic Protein: Comparison of Chondrichthye Basic Proteins with Those from Higher Vertebrates

Abstract: A basic protein has been purified from the CNS myelin of the gummy shark (Mustelus antarticus). Electroblotting was used to examine the capacity of rabbit antisera raised against this electrophoretically pure protein to recognize myelin basic protein from higher vertebrates. The antisera bound to two shark proteins including the original polypeptide antigen and to chicken, bovine, and human myelin basic proteins. Thus, the shark protein appeared to possess antigenic determinants that have been retained through evolutionary divergence of these proteins. Whereas bovine basic protein caused experimental allergic encephalomyelitis in guinea pigs, animals that received injections of the shark protein showed neither clinical nor histological signs of this disease. However, tests for delayed-type hypersensitivity and for Arthus reaction following injection with the shark protein revealed a T-cell-mediated response to this antigen and substantial cross-reactivity with higher vertebrate basic proteins. Analysis of the amino acid composition of the shark protein, and comparison of its tryptic peptide map with that of the bovine protein, revealed substantial changes in the amino acid sequence. Although the shark protein has some antigenic determinants in common with the proteins from higher vertebrates, it appears that much of the structure differs.     

Age-related changes in the development of experimental autoimmune encephalomyelitis

A prominent feature of multiple sclerosis is its high incidence of onset in the third decade of life and its relatively rare onset in persons older than 50 years. In order to study age-related restriction of clinical expression, a comparative biochemical, immunological and histological study was undertaken during development of experimental autoimmune encephalomyelitis (EAE) in young (7 weeks) and middle-aged (15 months) Wistar rats. Young rats showed characteristic clinical signs 12-16 days postinduction, and then they spontaneously recuperated. In middle-aged rats, the incidence of clinical signs was significantly reduced, with a later onset of the disease. Similar biochemical and histological alterations were detected in both age groups, but they were present in a later stage in middle-aged animals. However, cellular and humoral immune responses to myelin basic protein (MBP) were observed 15 days postinduction in all EAE animals. The study of anti-MBP IgG isotype pattern in 7-week-old animals indicated a predominant Th1-type immune response during the acute stage of EAE, with antibodies predominantly recognizing the MBP 96-128 peptide. In contrast, 15-month-old animals showed a less prominent Th1 response, without any epitope dominance. The changes in immune function found in middle-aged animals may account for the different susceptibility and expression of EAE, and may also be relevant to the different clinical expression observed in multiple sclerosis with maturation.     

Experimental allergic encephalomyelitis in the guinea pig: Variation in peripheral blood lymphocyte responsiveness to myelin basic protein during disease development

Peripheral blood lymphocytes (PBL) from guinea pigs with experimental allergic encephalomyelitis (EAE) induced by sensitization with bovine whole white matter, proliferated in vitro upon exposure to bovine myelin basic protein (B-MBP). The degree of the response increased with clinical severity. PBL from EAE-sensitized guinea pigs which failed to develop clinical disease did not respond to B-MBP. PBL from complete Freund's adjuvant-sensitized and nonsensitized normal guinea pigs were not responsive to B-MBP. EAE-sensitized animals displaying clinical signs of disease showed concanavalin A (Con A) responsiveness which paralleled that of B-MBP. Animals that did not develop EAE demonstrated Con A responses similar to those of control guinea pigs. Thus, in this acute autoimmune demyelinating condition, PBL responsiveness to B-MBP might provide a monitor of disease development.     

Suppression and reversal of allergic encephalomyelitis in guinea pigs with a non-encephalitogenic analogue of the tryptophan region of the myelin basic protein.

The administration of synthetic peptide S42 leads to suppression and reversal of experimental allergic encephalomyelitis (EAE) induced in guinea pigs by myelin basic protein. Peptide S42 contains a linear sequence of 21 amino acid residues, H-Phe-Ser-Trp-Gln-Lys-Phe-Ser-Trp-Gln-Lys-Phe-Ser-Trp-Gln-Lys-Phe-Ser-Trp-Gln-Lys-Gly-OH, made up of four repeating unit sequences of H-Phe-Ser-Trp-Gln-Lys-OH in addition to a C-terminal glycine. Injected at relatively high doses, peptide S42 is non-encephalitogenic. It induces delayed-type hypersensitivity which is not followed by EAE, and elicits delayed-type hypersensitivity responses in peptide S42, encephalitogenic trytophan peptide, or BP-challenged animals for either of the three antigens. The repeating unit sequence of peptide S42 is analogous to the encephalitogenic tryptophan region of the BP molecules . The sequence homology is responsible for cellular recognition of this antigen by the skin test assay and suggests in vivo interaction between peptide S42 and EAE-inducing cells leading to suppression and reversal of disease.     

Correlation between active rosette formation and delayed cutaneous hypersensitivity in experimental allergic encephalomyelitis

The effect of encephalitogenic myelin basic protein, BP, on active rosette-forming T cells (ARFC) was compared to that of nonencephalitogenic peptide S42, a synthetic analogue of the tryptophan region of BP. Depression of ARFC by these antigens was reversible within 24 h after a second dose of the antigen into the skin, or after in vitro incubation of lymphocytes with the sensitizing antigen (Ag-ARFC). The ratio of Ag-ARFC to ARFC rose with time following the sensitization but fell shortly before the clinical onset of experimental allergic encephalomyelitis in animals sensitized with BP. In contrast, the Ag-ARFC/ARFC ratios for animals sensitized with peptide S42 reached plateau levels from which they did not drop. The kinetics of the Ag-ARFC/ARFC responses paralleled those for delayed-type skin hypersensitivity (DTH) in the respective animals. The DTH responses rose following sensitization and fell shortly after the appearance of clinical signs of EAE. The results of this study provide in vitro and in vivo evidence for sensitization to myelin basic protein, and focus attention on the ARFC as a measure for an immunologically active cell population which may be quantitated by antigenic stimulation.Abbreviations used in this report EAE experimental allergic encephalomyelitis - DTH delayed-type skin hypersensitivity - ARFC active rosette-forming T cells - Ag-ARFC antigen-stimulated active rosette-forming T cells - TRFC total rosette-forming T cells     

Failure of myelin basic protein to prevent or suppress experimental allergic encephalomyelitis in guinea pigs

The encephalitogenic myelin basic protein (BP) was reported to be effective in preventing and suppressing the development of experimental allergic encephalomyelitis (EAE) when animals were treated before or after encephalitogenic challenge, respectively. In this report we show that pretreatment with 15 daily doses of 2.5 or 0.15 mg homologous BP (in IFA) failed to protect guinea pigs from subsequent challenge with encephalitogenic emulsion. Similarly, 15 daily injections of 1.0, 2.5, 5.0, or 10.0 mg guinea pig BP (in IFA) did not suppress development of or arrest ongoing EAE when the treatment was initiated on days 1, 4, 8, or 11 after an encephalitogenic challenge. The results show that over 50% of the treated animals developed hind leg paralysis (HLP), incontinence, or both, and the incidence of HLP was not altered significantly by a 10-fold increase in the amount of BP used for daily treatment. Further, all the treated and challenged animals developed histological lesions characteristic of disease. Treatment with BP delayed disease onset, prolonged the period of paralysis leading to recovery from HLP, and reduced both the prevelence of histological lesions as well as the incidence of death. It may be concluded that under these experimental conditions the administration of BP failed to protect from or suppress development of EAE.     

A synthetic peptide from myelin proteolipid protein induces experimental allergic encephalomyelitis

Immunization of animals with proteolipid protein, the major protein constituent of central nervous system myelin, produces experimental allergic encephalomyelitis. The goal of the present study was to identify an encephalitogenic determinant of this protein. For this purpose, SWR mice were immunized with five groups of pooled synthetic peptides corresponding to various regions of the myelin proteolipid protein sequence. Clinical EAE was observed in only one group. Inguinal lymph node cells from animals in this group responded ([3H]thymidine incorporation) to a peptide within the pool containing residues 103-116 YKTTICGKGLSATV. Mice subsequently immunized with 50 nmol of this peptide developed severe EAE within 3 wk, and their T cell-enriched inguinal lymph node cells responded specifically to this peptide. Control mice immunized to proteolipid peptide 202-217 DARMYGVLPWNAFPGK did not develop experimental allergic encephalomyelitis, and their inguinal lymph node cells were unresponsive to either peptide. Thus, a peptide corresponding to a sequence within the proteolipid protein can produce classical acute experimental allergic encephalomyelitis. This is the first report of a synthetic encephalitogenic peptide from myelin proteolipid protein.     

Prevention of experimental allergic encephalomyelitis by bacterial lipopolysaccharides: inhibition of cell-mediated immunity

The immunization of Lewis rats with bacterial lipopolysaccharides (LPS) precomplexed to guinea pig myelin basic protein (BP) in complete Freund's adjuvant inhibits the development of experimental allergic encephalomyelitis (EAE) in these animals. These protected animals fail to manifest significant in vivo delayed-type hypersensitivity skin tests and in vitro lymphocyte proliferative responses to BP. Our results indicated that LPS induces a nonspecific reduction in immune reactivity of BP in Lewis rats.     

Treatment of experimental allergic encephalomyelitis with an inhibitor of cathepsin D (pepstatin)

Intraperitoneal administration of pepstatin (2 mg/day for 5 weeks) to Lewis rats subjected to experimental allergic encephalomyelitis (EAE) (induced by guinea pig spinal cord and pertussis vaccine) suppressed the appearance of clinical signs of disease, and reduced the severity and incidence of CNS lesions normally associated with this disease. Administration of pepstatin for shorter periods to Lewis rats, or BSVS mice, or guinea pigs challenged with myelin basic protein delayed, but did not prevent clinical signs of EAE, but was accompanied in all cases by a less severe histopathology.     

Niridazole suppression of experimental allergic encephalomyelitis in Lewis rats.

Administration of niridazole to Lewis rats beginning 2 days before sensitization with guinea pig spinal cord in combination with immunologic adjuvants exerted a dose related suppressive influence on development of experimental allergic encephalomyelitis (EAE). A daily dose of 75 mg/kg completely prevented clinical neurologic signs as well as markedly suppressed occurrence of immunohistopathologic manifestations of this autoimmune disease. A higher daily dose level of niridazole, i.e., 100 mg/k, also inhibited EAE but was associated with neurotoxic manifestations.     

Suppression and reversal of allergic encephalomyelitis in rhesus monkeys with basic protein and peptides

We have extended earlier studies on the suppression of clinically evident experimental allergic encephalomyelitis (EAE) in monkeys, using repeated injections of human basic protein. The results confirm that after suppressive treatment, recovered animals remain clinically normal and do not show spontaneous recurrence of symptoms. However, recovered animals are susceptible to EAE upon renewed challenge, and they develop the disease more rapidly and more severely than after the initial challenge; resuppression is also accomplished in these cases by the same methods used previously. The present results indicate further that the basic protein or peptide T administered without mycobacteria is effective in suppressing the development of basic protein-induced EAE regardless of the species from which it was derived.     

Adoptive transfer of experimental allergic encephalomyelitis with activated spleen cells: Comparison of in vitro activation by concanavalin A and myelin basic protein

Adoptive transfer of experimental allergic encephalomyelitis (EAE) was carried out in Lewis rats using splenic lymphocytes incubated in vitro with either concanavalin A (Con A) or myelin basic protein (MBP). Requirements were established for sensitization of donors, culture conditions, numbers of transferred cells, and incubation period of EAE in recipients. These were strikingly similar whether Con A or MBP was used. In addition, cellular proliferation in vitro was not required in either system, but proliferation after transfer to the recipient was essential for the development of clinical signs and histological lesions. These methods have potential value for analyzing mechanisms of immune induction in this classic model of autoimmune disease.